Pulsatile Perfusion Bioreactor for Mimicking, Controlling, and Optimizing Blood Vessel Mechanics

ABSTRACT

A pulsatile perfusion bioreactor for culturing one or more engineered blood vessels having a lumen and a wall is provided. The bioreactor includes a chamber for holding the engineered blood vessel and cell culture media; a mechanical property monitoring system for measuring axial tensile stress and strain, circumferential tensile stress and strain, and/or shear stress imparted on the vessel wall; and a pump system for delivering cell culture media through the vessel lumen, wherein the vessel is exposed to a composite pressure waveform and a composite flow waveform as the media is delivered there through. The pump system includes a steady flow and peristaltic pumps. Further, the composite pressure and flow waveforms each include a mean component, a fundamental frequency component, and a second harmonic frequency component. The bioreactor also includes a computer interface for monitoring and adjusting the composite waveforms to maintain a predetermined stress levels.

RELATED APPLICATIONS

The present application claims priority to U.S. Provisional ApplicationSer. No. 62/143,844, filed on Apr. 7, 2015, which is incorporated hereinin its entirety by reference thereto.

BACKGROUND

The field of vascular tissue engineering seeks to generate functionalblood vessels with properties similar to that of healthy tissue.Engineered tissues may originate from biocompatible scaffolds embeddedwith diverse populations of cells or through altering existing tissuesexposed to controlled stimulation that initiate known intrinsic adaptiveprocesses. The demand for such functional tissues is especially great inthe vascular system, where engineered blood vessels (EBVs) could be usedto replace diseased or damaged blood vessels in patients suffering fromadvanced stage atherosclerosis or other focalized degenerative diseases.For example, nearly 400,000 coronary artery bypass grafts and around50,000 peripheral vascular grafts were performed in the United Stateseach year. However, patients often lack viable autograft tissue andpurely synthetic replacements (e.g., DACRON® grafts) become occludedwhen used to replace small diameter vessels. As such, there is a needfor the development of functional EBVs that are biocompatible, areanti-thrombogenic, and that exhibit autograft-like levels of burst andfatigue strength. Accordingly, various approaches have been employedover the years to engineer blood vessels that meet these requirements,yet few have reached advanced stage clinical trials. Therefore thepractical and commercial development of this technology remains anemerging field.

In the development of EBVs, the choice of scaffold material, cell type,and assembly are typically considered. The mechanical environment andthe bioreactor utilized for culturing the EBV must also be considered.The mechanical environment includes the biaxial stresses and stretchesgenerated in the circumferential and the axial directions of the EBV, aswell as the fluid-induced wall shear stresses focused along theendothelial cell (EC) lined lumen of the EBV. In vivo, thecircumferential loading is a result of pressurization and the axialloading is a result of somatic growth. It has also been observed thatnatural tissues seek to restore levels of mechanical stress. Forexample, in sustained hypertension, which elicits an acute increase incircumferential wall stress, the primary remodeling outcome is anincrease in wall thickness, which in turn acts to restore the wallstress to baseline, or normotensive, stress levels. Accordingly, themechanical environment has been identified as a major contributor to thegrowth and remodeling of a biomimetic EBV and is an important physicalfactor in vascular graft generation and homeostasis. Specifically,cyclic stretching of intramural vascular cells initiates proliferation,promotes the release of growth factors, alters fiber realignment,regulates smooth muscle cell (SMC) contractile phenotype, and encouragesoverall extracellular matrix (ECM) synthesis (e.g., collagen,tropoelastin, etc.) and tissue turnover by SMCs and fibroblasts.Similarly, the frequency, direction, and magnitude of shear stress onECs governs metabolic activities, nutrient exchange, cellularmorphology, stress fiber alignment, and SMC phenotype, and also controlparacrine factors including nitric oxide release, which is a majormediator of remodeling and homeostasis.

Over the last fifty years, vascular perfusion bioreactors have beendeveloped primarily as research tools but have recently become availablein the commercial market. However, commercially available bioreactorsare not designed to implement the comprehensive mechanical objectivesdiscussed above that are needed to create a truly biomimetic EBV. Assuch, a need exists for a EBV bioreactor that has the capability tooptimize mechanical (stress) objectives, which would, in turn, minimizeculture time and maximize output. Furthermore, a need exists for abioreactor specific for culturing EBVs that can impose and test biaxialloads, can deliver specific biomimetic pressure and flow profiles, canbe scalable for different vessels and animals, and can provide for realtime data collection and assessment. A need also exists for a bioreactorthat can be autoclaved, maintain sterility for prolonged culture times,promote nutrient and gas exchange, actively maintain temperature and pH,permit cell seeding, and allow for chemical stimulation and assessment.Such a device could be used in the commercial setting and not solely asa research tool.

Moreover, the ultimate success of an EBV lies in its ability to performin the intended environment. The biomimetic hemodynamic loads on the EBVare unique amongst species and anatomical location, thus the knowledgeand application of vessel-specific hemodynamics and axial loading thatmimic the intended graft condition are both crucial during tissuedevelopment in order to avoid hemodynamically-induced pathologies. Assuch, a need exists for a bioreactor that could prescribe dynamicpressure and flow waveforms during culture that incorporate complexphasic relationships that are not static or simply sinusoidal. In otherwords, a need exists for a bioreactor that can deliver variable stressesto an engineered blood vessel through the application and control ofdynamic pressure and flow waveforms so that the engineered blood vesselcan be conditioned and remodeled during ex-vivo culture so that itultimately possesses properties that mimic a native blood vessel.

SUMMARY OF THE INVENTION

The present invention is directed to a pulsatile perfusion bioreactorfor culturing one or more engineered blood vessels having a lumen and awall. This invention also provides mechanomimetic stimulation to theblood vessels and can be used to study physiological dependent vascularprocesses such as the response to endovascular interventions andpharmaceutical administration. The pulsatile perfusion bioreactorincludes one or more chambers for holding the engineered blood vesseland cell culture media; a mechanical property monitoring system formeasuring axial tensile stress, circumferential tensile stress, shearstress, or a combination thereof imparted on the wall of the engineeredblood vessel and for measuring and controlling axial stretch,circumferential stretch, or a combination thereof imparted on the wallof the engineered blood vessel; and a pump system for delivering cellculture media through the lumen of the engineered blood vessel. The pumpsystem includes a steady flow pump and a peristaltic pump such that whencultured inside the pulsatile perfusion bioreactor, the engineered bloodvessels are exposed to a composite pressure waveform and a compositeflow waveform as the cell culture media is delivered through the lumen.The composite pressure waveform comprises a mean pressure component, afundamental frequency (or first harmonic) pressure component and asecond harmonic frequency pressure component, while the composite flowwaveform component comprises a mean flow component, a fundamentalfrequency (or first harmonic) flow component and a second harmonicfrequency flow component. However, it is to be understood thatadditional harmonic flow frequencies can be added to improve resolution.The pulsatile perfusion bioreactor further includes a computer interfacefor monitoring and adjusting the composite pressure waveform, thecomposite flow waveform, or a combination thereof to maintain apredetermined axial tensile stress level, a predeterminedcircumferential stress level, a predetermined shear stress level, or acombination thereof.

In an additional embodiment, the composite pressure waveform and thecomposite flow waveform can be derived from a pressure waveform and aflow waveform of a native blood vessel, and the engineered blood vesselcan be a replacement for the native blood vessel.

In another embodiment, the steady flow pump can deliver the meanpressure component of the composite pressure waveform and the mean flowcomponent of the composite flow waveform. In still another embodiment,the peristaltic pump can deliver a pulsatile flow of cell culture mediathrough the lumen via a first pump head and a second pump head. Forexample, the first pump head can provide the fundamental frequencypressure component of the composite pressure waveform and thefundamental frequency flow component of the composite flow waveform,while the second pump head can provide the second harmonic frequencypressure component of the composite pressure waveform and the secondharmonic frequency flow component of the composite flow waveform. In afurther embodiment, the peristaltic pump can also include a third pumphead, where the third pump head can provide a third harmonic frequencypressure component of the composite pressure waveform and a thirdharmonic frequency flow component of the composite flow waveform.

In yet another embodiment, the bioreactor can also include a compliancechamber. In one more embodiment, the bioreactor can include a pressuretransducer. In an additional embodiment, the bioreactor can also includea stepper motor controlled pinch valve. In one particular embodiment,the bioreactor can also include a camera for measuring the engineeredblood vessel length, wall diameter, wall thickness, or a combinationthereof.

In one embodiment, the bioreactor chamber can be located in anincubator. Further, the engineered blood vessel can include a naturalmaterial or a synthetic material. Moreover, the engineered blood vesselcan include endothelial cells, smooth muscle cells, or a combinationthereof.

In yet another embodiment, the present invention is directed to a methodof culturing one or more engineered blood vessels having a lumen and awall inside a pulsatile perfusion bioreactor. The method includesinserting the engineered blood vessel to be cultured into a chamber;filling the chamber with cell culture media; delivering cell culturemedia through the lumen of the engineered blood vessel via a pumpsystem, wherein the engineered blood vessel is exposed to a compositepressure waveform and a composite flow waveform as the cell culturemedia is delivered through the lumen, the pump system comprising asteady flow pump and a peristaltic pump, wherein the composite pressurewaveform comprises a mean pressure component, a fundamental frequencypressure component and a second harmonic frequency pressure component,and wherein the composite flow waveform component comprises a mean flowcomponent, a fundamental frequency flow component, and a second harmonicfrequency flow component; measuring axial tensile stress,circumferential tensile stress, shear stress, axial stretch,circumferential stretch, or a combination thereof imparted on the wallof the engineered blood vessel via a mechanical property monitoringsystem; and monitoring and adjusting the composite pressure waveform,the composite flow waveform, or a combination thereof to maintain apredetermined axial tensile stress level, a predeterminedcircumferential stress level, a predetermined shear stress level, apredetermined axial stretch level, a predetermined circumferentialstretch level, or a combination thereof via a computer interface.

In an additional embodiment, the composite pressure waveform and thecomposite flow waveform can be derived from a pressure waveform and aflow waveform of a native blood vessel, and the engineered blood vesselcan be a replacement for the native blood vessel.

In another embodiment, the steady flow pump can deliver the meanpressure component of the composite pressure waveform and the mean flowcomponent of the composite flow waveform.

In still another embodiment, the peristaltic pump can deliver apulsatile flow of cell culture media through the lumen. The peristalticpump can include a first pump head and a second pump head, where thefirst pump head can provide the fundamental frequency pressure componentof the composite pressure waveform and the fundamental frequency flowcomponent of the composite flow waveform, while the second pump head canprovide the second harmonic frequency pressure component of thecomposite pressure waveform and the second harmonic frequency flowcomponent of the composite flow waveform.

In yet another embodiment, the peristaltic pump can further include athird pump head, where the third pump head provides a third harmonicfrequency pressure component of the composite pressure waveform and athird harmonic frequency flow component of the composite flow waveform.

In an additional embodiment, the pulsatile perfusion bioreactor caninclude a compliance chamber, where the compliance chamber canfacilitate adjustment of the composite pressure waveform and can reducenoise caused by the peristaltic pump. In still another embodiment,pressure can be measured via a pressure transducer and a stepper motorcontrolled pinch valve can be utilized to adjust resistance within thepulsatile perfusion bioreactor, wherein adjusting the resistance resultsin an adjustment to the pressure.

Other features and aspects of the present invention are set forth ingreater detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

A full and enabling disclosure of the present invention, including thebest mode thereof to one skilled in the art, is set forth moreparticularly in the remainder of the specification, which includesreference to the accompanying figures.

FIG. 1 shows an example of the physiological blood pressure (P) and flow(Q) waveforms that can be created an applied to an EBV via the pulsatileperfusion bioreactor of the present invention. This example mimics theconditions of a human radial artery.

FIG. 2 shows a schematic of one embodiment of the pulsatile perfusionbioreactor of the present invention.

FIG. 3a shows a porcine renal artery cultured for 10 days in thepulsatile perfusion bioreactor of the present invention prior toadministration of phenylephrine to elicit smooth muscle cell (SMC)contraction.

FIG. 3b shows a porcine renal artery cultured for 10 days in thepulsatile perfusion bioreactor of the present invention afteradministration of phenylephrine to elicit smooth muscle cell (SMC)contraction.

FIG. 4 shows the 4-element Windkessel model as an electro-hydraulicanalogy with two resistors, an inductor, and a capacitor, where thelocation of the blood vessel for culturing is marked with an (x).

FIG. 5a shows the composite hemodynamic pressure waveform (dotted line)and the mean hemodynamic pressure waveform (n=0; solid line), as well asthe fundamental frequency (or first harmonic frequency) component (n=1,open circle waveform), and two harmonic frequency components (n=2, opensquare waveform and n=3, open diamond waveform) of the compositehemodynamic pressure waveform, from a pig renal artery.

FIG. 5b shows the composite hemodynamic flow waveform (dotted line) andthe mean hemodynamic flow waveform (n=0; solid line), as well as thefundamental frequency (or first harmonic frequency) component (n=1, opencircle waveform), and two harmonic frequency components (n=2, opensquare waveform and n=3, open diamond waveform) of the compositehemodynamic flow waveform, from a pig renal artery.

FIGS. 6a-6c show the velocity profiles (3D map) and measured centerlinevelocity (dotted line) for (a) a Human Radial Artery, (b) a Pig RenalArtery, (c) and a Mouse Aorta.

FIGS. 7a-7c show the mean (solid flat line) and pulsatile (solidparabolic/sinusoidal line) wall shear stress (τ_(w),) for (a) a HumanRadial Artery, (b) a Pig Renal Artery, and (c) a Mouse Aorta.

FIGS. 8a-8c show the desired (solid line) and simulated (open circles)pressure responses using the best fit parameters of theelectro-hydraulic 4-element Windkessel model. The desired pressure isreported in the literature for (a) a Human Radial Artery, (b) a PigRenal Artery, (c) and a Mouse Aorta which also shows a zoomed-in sectionon one mouse cardiac cycle.

Repeat use of reference characters in the present specification anddrawings is intended to represent the same or analogous features orelements of the present invention.

DETAILED DESCRIPTION

Reference now will be made to the embodiments of the invention, one ormore examples of which are set forth below. Each example is provided byway of an explanation of the invention, not as a limitation of theinvention. In fact, it will be apparent to those skilled in the art thatvarious modifications and variations can be made in the inventionwithout departing from the scope or spirit of the invention. Forinstance, features illustrated or described as one embodiment can beused on another embodiment to yield still a further embodiment. Thus, itis intended that the present invention cover such modifications andvariations as come within the scope of the appended claims and theirequivalents. It is to be understood by one of ordinary skill in the artthat the present discussion is a description of exemplary embodimentsonly, and is not intended as limiting the broader aspects of the presentinvention, which broader aspects are embodied exemplary constructions.

It is understood that mechanical signals are key mediators of thecellular processes which underlie blood vessel remodeling, includingproliferation, differentiation, migration, and protein synthesis.Further, blood vessels are dynamic in form, function, and pulsatility(i.e., the difference between the maximum and minimum blood pressure andflow). Blood pressure induces periodic circumferential stresses, whichare sensed by smooth muscle cells and fibroblasts embedded in thevascular wall, whereas blood flow induces periodic and often oscillatorywall shear stresses, which are sensed by endothelial cells lining thelumen. In addition, it has been shown that the production ofextracellular matrix material and the alignment of smooth muscle cellsand fibroblasts both depend on the rate and magnitude of cyclicstretching. Furthermore, gene profiles of endothelial cells in cultureare dependent upon the magnitude and frequency of flow oscillation witha morphology that discriminates between different flow environments.Vascular pathologies also depend on the cyclic and oscillatory flow suchas those observed in atherosclerosis formation. Thus, blood vesselgrowth and remodeling are highly dependent on the pressure and flowwaveforms and stresses to which the blood vessel is exposed. As such, anaccurate means of applying pressure and flow waveforms to a EBV andadjusting the pressure and flow waveforms to achieve desired stress andloading objectives is important in obtaining a truly biomimetic EBV andcan be accomplished via the pulsatile perfusion bioreactor of thepresent invention.

Meanwhile, although in-vivo and ex-vivo experimental studies have beenconducted to delineate remodeling outcomes under prescribed loadingconditions, with a manifest dependence on vascular wall stresses, noex-vivo culture systems are available that can accurately translateremodeling principles (i.e., stress-driven growth) into the developmentof commercially available EBVs, where the amount of stress applied tothe EBV can be accurately controlled during culturing so that theengineered blood vessel is exposed to stresses equivalent to those towhich a native blood vessel is exposed. This is due, in part, to thecomplexity of conferring biomimetic pulsatile loading in athree-dimensional tissue culture environment that integrates both thefluid and solid domains, the challenges of retaining tissue viabilityfor extended culture periods, and the wide variation in the types andsizes of EBVs to be cultured. Furthermore, stress levels imparted to anEBV must be tightly controlled throughout culture, as exceeding materialand maturation state-specific stress thresholds can lead to materialfailure or potentially adverse cellular responses (i.e., apoptosis). Tosolve this problem, the pulsatile perfusion bioreactor of the presentinvention can accurately replicate the pressure and flow waveforms ofthe native vessel that the EBV is intended to replace and can make realtime adjustments to the waveforms as the EBV is cultured to maintaindesired stress levels. As such, the present inventors have found that anEBV cultured in the pulsatile perfusion bioreactor of the presentinvention can undergo remodeled during culture so that the end productmimics the native vessel it is to replace.

As discussed in more detail below, the present inventors have developedan ex-vivo pulsatile perfusion bioreactor that enables for the controlof mechanically-induced tissue growth and remodeling of an EBV thatclosely mimics in vivo conditions via the application of specifiedstress levels through the control of pressure and flow waveforms appliedto the EBV in culture. In other words, the pulsatile perfusionbioreactor of the present invention can control deterministic aspects ofblood vessel mechanics and can facilitate functional vascular tissueremodeling in engineered blood vessels having a synthetic or biologicalorigin. For instance, through application of biomimetic pressure andflow waveforms and real-time assessment of sample geometry andmechanical properties, the pulsatile perfusion bioreactor of the presentinvention enables the control and optimization of culture conditions forvarious sizes and types of engineered vascular tissues formed from bothnatural and synthetic materials. Further, the pulsatile perfusionbioreactor of the present invention allows for collection of sample datausing a computer interface with minimal human intervention, thusmitigating contamination potential and enabling a feedback controlsystem for optimizing vascular tissue growth. Dynamic flow-induced wallshear stress and three-dimensional tensile wall stress conditions can becontrolled and adjusted to predetermined objective values based on thetype of native blood vessel being replaced with the EBV, andflow/pressure profiles and imposed axial stretch can be tuned andadjusted during culture to maintain the predetermined objective values,resulting in a EBV having properties that mimic those of the nativeblood vessel that it will replace. Furthermore, the mechanicalsuitability of EBV can be tested directly within the pulsatile perfusionbioreactor of the present invention, which can streamline the iterativerefinement of culture protocol that is implicit in the development ofany tissue-engineered product.

In addition, while currently available bioreactors enable specificationof the global mechanical environment of vascular tissue samples (e.g.,lumen pressure, flow rate, and axial extension), the pulsatile perfusionbioreactor of the present invention additionally allows forspecification of the local mechanical environment of resident vascularcells (e.g., wall tensile stresses (circumferential and axial) andflow-induced shear stress). This is a critical distinction, as the localmechanical environment changes as the vessel remodels and is what drivesmechanosensitive cellular processes. The pulsatile perfusion bioreactorof the present invention can thus consistently impart biomimeticstresses to the EBV being cultured, and that tissue viability andfunction can be retained at least over a two-week culture period.Irrespective of the particular application, the pulsatile perfusionbioreactor of the present invention is configured to allow for theautomated control of fundamental mechanical signals governing vasculartissue remodeling. The driving mechanical stimuli for native vasculartissue growth and remodeling are wall stresses operative at the intima(e.g., flow-induced shear stress, which is sensed and regulated byvascular endothelial cells) and within the media (e.g., tensile stressin the axial and circumferential directions, which is sensed andregulated by vascular smooth muscle cells) of a blood vessel. Thus, theability to control these same mechanical quantities in a bioreactor canelicit control of growth and remodeling processes in cell-laden EBVs. Inorder to promote the cellular processes implicit in vascular tissuegeneration, it is critical that imposed stresses are derived frombiomimetic mechanical loading profiles (i.e., physiological pressure andflow waveforms). The application of biomimetic mechanical loading on theEBV culture in the pulsatile perfusion bioreactor of the presentinvention via the application of pressure and flow waveforms that mimicphysiological pressure and flow waveforms can thus create an ex-vivoenvironment in which vascular cell behavior can be understood,predicted, and ultimately directed towards functional engineered tissuegeneration.

As mentioned above, currently available bioreactor systems featurecontrol of the lumen pressure, flow rate, and in some cases the degreeof axial stretch to which samples are subjected, (i.e., control of theglobal mechanical loading). Further, such bioreactors utilize pressureand flow waveforms that are gross approximations of physiologic loading(e.g., sine waves or steady flows) such that the resulting mechanicalenvironment in which an EBV is cultured substantially deviates from thatof native vessels. In contrast, the pulsatile perfusion bioreactor ofthe present invention allows for user control of the wall stressimparted on the EBV, and thus imparts the user with the ability tocontrol of the local mechanical environment which drives key cellularprocesses. Such control is due to the use and application of pressureand flow waveforms that replicate native hemodynamics.

In order to control the local mechanical environment via the pulsatileperfusion bioreactor of the present invention, predetermined wall stressvalues can be programmed and automatically maintained through a feedbackloop that integrates mechanical and geometrical information obtainedthrough measurements performed on the EBV by a mechanical propertymonitoring system that is a component of the pulsatile perfusionbioreactor. Thus, as the EBV changes dimensions and/or mechanicalproperties as a consequence of the progression of the growth andremodeling processes, the loading imposed on the EBV by the pulsatileperfusion bioreactor system can be automatically tuned such thatpredetermined stress values are maintained. Thus, the pulsatileperfusion bioreactor of the present invention is configured to controland optimize the stimuli-response mechanisms which underpin vasculartissue growth. The various components of the pulsatile bioreactor of thepresent invention are discussed in more detail below.

Applied Pressure and Flow Waveforms

Before the pulsatile perfusion bioreactor system of the presentinvention can be programmed and used for the culture of an engineeredblood vessel, the pressure and flow waveforms to be applied duringculture to achieve the desired stress or loading levels must bedetermined. It is known that the shape, peak, minimum, and averagevalues of blood pressure and flow vary greatly amongst various vasculartissues. Similarly, hemodynamic and geometric differences exist betweendifferent mammalian species, even when comparing similar anatomicallocations (e.g., the ascending aorta). These waveforms are modified bythe different downstream reflections, the relative size of blood toblood vessel, and most notably the diversity in heart rate. Accordingly,pressure and flow waveforms often do not have the same shape and cannotbe accurately modeled as simple sine waves. Thus, these hemodynamicwaveforms have been quantified using the mean, fundamental (firstharmonic), and additional harmonic components of a Fourier seriesrepresentation. Further, the electro-hydraulic analogy provides aconvenient methodology enabling a translation of periodic hemodynamicqualities into electrical circuits where Fourier analysis iscommonplace. Here the voltage, current, capacitance, resistance, andinductance are represented as pressure, flow rate, compliance,resistance and inertia. The simplicity of this technique makes it wellsuited for the practicality of the laboratory bench as physicalstructures are represented by pumps, valves, compliance chambers, andculture media.

As shown in FIG. 1, the typical pressure (P) and flow (Q) waveforms fora blood vessel are complex and are not simple sine waves or steadyflows. After the pressure and flow waveforms similar to those shown inFIG. 1 are determined for the specific native vessel to be replicated asa EBV in order to achieve desired stress levels to promote the cultureof the EBV in a biomimetic environment, the waveforms are converted intomathematical expressions that can be applied to the EBV positionedinside the pulsatile perfusion bioreactor of the present invention via acomputer interface and specific hardware (e.g., a compliance chamber,pinch valve, etc.).

Specifically, the present inventors have taken the pressure and flowprofiles for various types of blood vessels in their native environmentand have then converted those waveforms to mathematical models viaFourier Transform, where the mathematical models are then used torecreate the pulsatile flow and pressure waveforms experienced by anative vessel during the culture of a EBV in the pulsatile perfusionbioreactor of the present invention. Such a determination can be basedon Womersley's approach, which uses a periodic pressure gradient topredict the temporal velocity profile within a vessel. This profile doesnot follow a constant parabolic shape throughout the cardiac cycle, canexhibit flow reversal, and may have a maximum velocity that is notnecessarily at the center of the lumen of the vessel. However,centerline velocity can be easily measured and can be performednon-invasively with modern approaches such as vascular Doppler, so thepresent inventors have utilized this approach. Further, to calculateother hemodynamic variables of interest, namely volumetric flow rates,velocity profiles, and wall shear stresses from the measured centerlinevelocity, the present inventors have utilized an inverse Womersleyapproach to provide both temporal and spatial blood flow quantities forvarious types of blood vessels for which replacement EBVs can becultured utilizing the pulsatile perfusion bioreactor of the presentinvention.

As a result of the transformation of the pressure and flow waveformsinto mathematical expressions, multiple waveforms can be applied to theEBV, resulting in a composite pressure waveform and a composite flowwaveform. Each composite waveform can include a mean pressure component,a fundamental frequency component (e.g., a first harmonic frequencycomponent), and additional harmonic frequency components. While in thepast, literature has indicated that a fundamental frequency componentplus four additional harmonic frequency components are required wasrequired to accurately replicated the pressure and flow waveforms towhich a native blood vessel is exposed, the present inventors havesurprisingly found that an accurate model of composite pressure and flowwaveforms and be developed utilizing less than four additional harmonicfrequency components. For instance, in one particular embodiment, thecomposite pressure and flow waveforms includes a mean or steadycomponent, a first harmonic frequency component (also referred to as thefundamental frequency component), and a second harmonic frequencycomponent. In a further embodiment, the composite pressure and flowwaveforms can include a third harmonic frequency component. Moreover,although not required, additional harmonic frequency components can beemployed in the composite pressure and flow waveforms to achievecomposite waveforms that most closely mimic the waveforms to which thenative blood vessel of interest is exposed, resulting in the EBV beingexposed to biomimetic tensile and shear stresses. In other words, it isto be understood that any suitable number of harmonic frequencies can beutilized up to an n^(th) harmonic frequency, where n is a whole numberthat is 2 or greater.

One process for deriving the composite pressure and flow waveforms thatcan be programmed into a computer interface is described in more detailbelow in Example 1.

Bioreactor Components

Once the waveforms to be applied to the EBV are determined, the processfor culturing the EBV in the pulsatile perfusion bioreactor of thepresent invention can be initiated. Referring to FIG. 2, in oneembodiment of the present invention, the pulsatile perfusion bioreactor100 includes a computer interface 101 that controls a pump system thatcan include a steady flow pump 102 that can deliver a mean flow andpressure waveform 103 and a peristaltic pump 104, which can includemultiple pump heads depending on the number of harmonic frequencycomponents to deliver as part of the composite pressure and flowwaveforms. For instance, as shown in FIG. 2, the peristaltic pump caninclude a first pump head 106, a second pump head 108, and a third pumphead 110 to deliver a first harmonic frequency component 112 (i.e., thefundamental frequency component), a second harmonic frequency component114, and, optionally, a third harmonic frequency component 116.Additional harmonic frequency components of the composite waveforms canbe incorporated into the pump system via additional pump heads.

In one particular embodiment of the pulsatile perfusion bioreactor 100of the present invention, the computer interface 101 is programmed suchthat cell culture media is introduced via the pump system into the lumenof an engineered blood vessel (EBV) 128 being cultured in a bioreactorchamber 126 positioned inside an incubator 124, such that the EBV 128 issubjected to the composite pressure waveform and the composite flowwaveform from the programmed mean flow 103 and harmonic frequencywaveforms 112, 114, and 116. The EBV 128 is positioned in the bioreactorchamber 126, which is filled with cell culture media 130, where themedia can be introduced and replaced via media exchange port 140. Inaddition, media is delivered through the proximal end 136 of the EBV 128in a pulsatile manner via inlet tubing 118 in the form of the compositeflow waveform discussed above and can then be circulated through thelumen of the EBV 128 and returned to the pump system via outlet tubing119. The composite waveforms discussed above can be applied to the EBV128 and monitored and adjusted, along with axial wall stretch, via afeedback loop as growth and remodeling of the EBV 128 progresses tomaintain the desired level of tensile and shear wall stresses on theEBV. For instance, utilizing measurements recorded via a mechanicalproperty monitoring system 134, which can include tensile testingcomponents, a camera, etc. (not shown), a stepper motor controlled pinchvalve 132 located at a distal end 140 of the EBV 128 can be adjusted tocontrol the pressure applied to the EBV 128 as tracked via a pressuretransducer 120 integrated into the pulsatile perfusion bioreactor 100,while a compliance chamber 120 can also be used to make adjustments tothe pulsatility of the applied waveforms. The mechanical propertymonitoring system 134 can also include a force transducer to measurestatic and dynamic axial forces. From these forces, a real-timeassessment of the static and dynamic stresses can be calculated.Further, a pair of electronic linear stages (not shown) can be used toapply static or dynamic axial stretching to the EBV as desired.

The aforementioned pulsatile perfusion bioreactor 100 can successfullyculture a EBV 128 over an extend period of time, such as up to about 10days, as shown via a comparison of FIGS. 3a and 3b . For instance, FIG.3a shows a porcine renal artery 128 a cultured for 10 days in thepulsatile perfusion bioreactor of the present invention prior toadministration of phenylephrine to elicit smooth muscle cell (SMC)contraction. FIG. 3b shows a porcine renal artery 128 b cultured for 10days in the pulsatile perfusion bioreactor of the present inventionafter administration of phenylephrine to elicit smooth muscle cell (SMC)contraction. The ability of the porcine renal artery 128 b to exhibitSMC contraction and EC dependent dilations demonstrates that thepulsatile perfusion bioreactor of the present invention can culture anEBV and demonstrate cell function afterwards. The present invention maybe better understood with reference to the following example(s).

EXAMPLE 1

Example 1 demonstrates the ability to determine the pressure and flowwaveforms for various types of blood vessels in their nativeenvironment, which can then be converted to mathematical models viaFourier Transform, where the mathematical models can be used to recreatethe pulsatile pressure and flow waveforms to which a native vessel isexposed during the culture of the EBVs of the present invention. Variouswaveform components can be combined to create composite pressure andflow waveforms to apply to the EBV via the pump system described above.

Introduction

In Example 1, the groundwork for recreating native blood vesselhemodynamics for a EBV undergoing tissue culture in a pulsatileperfusion bioreactor is achieved by recreating the blood pressure andcenterline flow velocity waveforms described in the classic andcontemporary literature. These waveforms are digitized and representedas mathematical equations using a Fourier series reconstruction. Thevolumetric flow rates, found using an inverse Womersley approach, areused as the input into a 4-Element Windkessel electro-hydraulic analogywith best fit parameters that provide a basis for the design of theculture system components to match in vivo blood pressures. Thetime-dependent wall shear stresses and velocity profiles are calculatedand compared from the literature sources providing groundwork for theidentification of key hemodynamic features enabling intra- andinter-species comparisons.

Materials and Methods Pulsatile Hemodynamics

Arterial hemodynamics are characterized by cyclic pulsatility of bothpressure and flow waveforms, the magnitude and behavior of which dependon cardiac contractility, the blood vessel location, and downstreamresistances. In contrast, venous system hemodynamics consistpredominantly of steady blood pressure and flow components. The in vivo,pulsatile, volumetric flow rate q(t) and wall shear stress τ_(w)(t) caneasily be calculated when the temporal velocity profile u(r,t) is knownusing

$\begin{matrix}{{{q(t)} = {2\pi {\int_{0}^{r_{a}}{{{ru}\left( {r,t} \right)}\ {r}}}}}{and}} & (1) \\{{\tau_{w}(t)} = {{- \mu}{\frac{\partial{u\left( {r_{a},t} \right)}}{\partial r}.}}} & (2)\end{matrix}$

Here r is the radial location within the lumen of the vessel with fixedinner wall radii r_(a) and μ is the viscosity of blood assumed to beconstant at high shear rates.

From the momentum balance of Navier-Stokes, the governing equation forunsteady, axisymmetric laminar flow in a rigid tube, where gravitationaleffects are neglected is solved using the Womersley approach. Thenon-dimensional Womersley number to relates pulsatility to viscouseffects at that frequency, so that

α_(n) =r _(a)(ω_(n)ρ/μ)^(1/2)   (3)

where ρ is the density of blood and ω_(n) represents the heartbeatfrequency with n=1 the fundamental frequency and n=2,3 the subsequentharmonics in rad/s.

Normally the entire velocity profile is unknown and only the centerlinevelocity u(0,t) profile is the only value reported (e.g., from pulsedwave Doppler). The complete velocity profile can be found from

$\begin{matrix}{{u\left( {r,t} \right)} = {{u_{s}\left( {1 - \frac{r^{2}}{r_{a}^{2}}} \right)} + {\sum\limits_{n = 1}^{3}\; {{u_{n}(t)}\left( \frac{{J_{0}\left( {i^{3/2}\alpha_{n}} \right)} - {J_{0}\left( {i^{3/2}\alpha_{n}{r/r_{a}}} \right)}}{{J_{0}\left( {i^{3/2}\alpha_{n}} \right)} - 1} \right)}}}} & (4)\end{matrix}$

where u_(s) and u_(n)(t) represent the steady and unsteady centerlinevelocities at each harmonic and where J₀ and J₁ are the Bessel functionsof the first kind of zeroth and first order respectively. The volumetricflow rate can be found from the centerline velocity using equation (1)and (4)

$\begin{matrix}{{q(t)} = {\pi \; {r_{a}^{2}\left( {\frac{u_{s}}{2} + {\sum\limits_{n = 1}^{3}\; {{u_{n}(t)}\left( \frac{{i^{3/2}\alpha_{n}{J_{0}\left( {i^{3/2}\alpha_{n}} \right)}} - {2\; {J_{1}\left( {i^{3/2}\alpha_{n}} \right)}}}{{i^{3/2}\alpha_{n}{J_{0}\left( {i^{3/2}\alpha_{n}} \right)}} - {i^{3/2}\alpha_{n}}} \right)}}} \right)}}} & (5)\end{matrix}$

Further, the time dependent wall shear stress at the wall can be foundvia

$\begin{matrix}{{\tau_{w}(t)} = {{- \frac{\mu}{r_{a}}}{\left( {{{- 2}\; u_{s}} + {\sum\limits_{n = 1}^{3}\; {{u_{n}(t)}\left( \frac{i^{3/2}\alpha_{n}{J_{1}\left( {i^{3/2}\alpha_{n}} \right)}}{{J_{0}\left( {i^{3/2}\alpha_{n}} \right)} - 1} \right)}}} \right).}}} & (6)\end{matrix}$

Blood pressure (via catheterization) and flow waveforms are convenientlyrepresented using a Fourier series expansion so that

q(t)=Σ_(n=0) ³ M _(n) ^(q) cos(ω_(n) t+φ _(n) ^(q))   (7)

and

p(t)=Σ_(n=0) ³ M _(n) ^(p) cos(ω_(n) t+φ _(n) ^(p)),   (8)

where M_(n) and φ_(n) are the magnitude and phase of pressure or flowwaves at each frequency. Of note, blood pressure and flow have the sameω_(n) but are out of phase.

When volumetric flow is given in the form of equation (7) instead of thecenterline velocity, the wall shear stress can also be calculateddirectly using the inverse Womersley approach, so that

$\begin{matrix}{{\tau_{w}(t)} = {\frac{\mu}{\pi \; r_{a}^{3}}\left( {{4\; M_{0}^{q}} - {\sum\limits_{n = 1}^{3}\; {M_{n}^{q}{\cos \left( {{\omega_{n}t} + \varphi_{n}^{q}} \right)}\left( \frac{i^{3}\alpha_{n}^{2}{J_{1}\left( {i^{3/2}\alpha_{n}} \right)}}{{i^{3/2}\alpha_{n}{J_{0}\left( {i^{3/2}\alpha_{n}} \right)}} - {2\; {J_{1}\left( {i^{3/2}\alpha_{n}} \right)}}} \right)}}} \right)}} & (9)\end{matrix}$

and is sometimes the case in the literature. Equation (9) is convenientas it allows for a calculation of wall shear stress in a cultured vesselbased on the magnitude of steady and pulsatile flow channels of thedevice described herein. These are described in the following section.

Electrical-Fluid Component Design

The electro-hydraulic analogy employed by earlier researchers provides abasis for a lumped parameter estimation to recreate in-vivohemodynamics. A pictorial representation of the electrical circuit withcorresponding hydraulic elements is shown in FIG. 3. Application ofKirchhoff s current (volumetric flow) law yields two first order,linear, differential equations:

$\begin{matrix}{{\frac{{p_{1}(t)}}{t} = {\frac{q(t)}{C} - \frac{p_{1}(t)}{R_{1}C}}}{and}} & (10) \\{\frac{{p_{2}(t)}}{t} = {{R_{2}\frac{{q(t)}}{t}} - \frac{R_{2}{p_{2}(t)}}{L}}} & (11)\end{matrix}$

where p₁(t) and p₂(t) are the pressures across resistors R₁ (mmHg·s/ml)and R₂ (mmHg·s/ml) respectively, C (ml/mmHg) is the downstreamcompliance, and L (mmHg·s²/ml) is the inductance. The volumetric flowrate represents the periodic displacement of a pump or pumps and isconsidered a configurable value in the pulsatile perfusion bioreactor ofthe present invention. Using Kirchoff's voltage (pressure) law, a thirdgoverning equation is obtained:

p(t)=p ₁(t)+p ₂(t)   (12)

with p(t) representing the pressure at the vessel culture location. Apractical interpretation of these variables as they pertain to aphysical design is provided in the results section.

Taking the Laplace transform of equations (10) to (12) provides atransfer function relating the volumetric flow input Q(s) to pressureoutput P(s) in the Laplace domain:

$\begin{matrix}{\frac{P(s)}{Q(s)} = \frac{{R_{1}R_{2}{CLs}^{2}} + {\left( {{R_{1}L} + {R_{2}L}} \right)s} + {R_{1}R_{2}}}{\left( {{{sR}_{1}C} + 1} \right)\left( {{sL} + R_{2}} \right)}} & (13)\end{matrix}$

The time response of the system described by equation (13) is simulatedusing the MATLAB® (Mathworks, Natick Mass.) function lsim.

Resistance, capacitance, and inductance values were found by minimizingthe error e_(P) between the desired pressure waveform and the systemmodeled in (13)

$\begin{matrix}{e_{P} = \sqrt{\frac{\sum\limits_{j = 1}^{S}\; {{p_{j} - p_{j}^{m}}}}{\sum\limits_{j = 1}^{S}\; p_{j}}}} & (14)\end{matrix}$

where j is the sample number of S total samples. The MATLAB® functionfminsearchbnd was used to achieve this minimization with lower limits ofzero for each variable. Defining e_(p) in this manner will generate alarge error when the desired and modeled pressures are out of phase.

Results

Combining the techniques of Fourier analysis and the inverse Womersleyapproach, key hemodynamic properties are discovered from in vivomeasurements of centerline velocity. Namely, temporal and spatialvelocity profiles are found and plotted as well as the temporalvolumetric flow and wall shear stress. From this information, theparameters of a blood vessel culture system can be designed toadequately recreate pulsatile and steady physiologic blood-pressure andflow in organ culture using an electro-hydraulic analogy of the4-element Windkessel model.

The results of a Fourier analysis of the pressure waveforms are shownfor a diverse set of 3 blood vessels in Table 1. The various resultspertain to the Human Radial Artery (H-RaA), the Human Renal Artery(H-ReA), and the Mouse Aorta (M-AoA).

TABLE 1 Magnitude and phase of blood pressure and flow for each harmonicfrequency of the heart rate for the Human Radial Artery (H-RaA), PigRenal Artery (P-ReA), and Mouse Aorta (M-AoA), as well as thedimensionless Womersley number at each frequency for the vessel. HeartM₀ ^(p) . . . M₃ ^(p) φ₀ ^(p) . . . φ₃ ^(p) M₀ ^(q) . . . M₃ ^(q) φ₀^(q) . . . φ₃ ^(q) α₀ . . . α₃ Rate (Hz) (mmHg) (rad) (ml/s) (rad) (..)H-RaA — 94.1 — 0.19 — — ID = 2.33 mm 7.87 12.8 2.81 0.05 1.75 1.64 15.78.05 −2.05 0.05 −2.30 2.31 23.6 4.14 −0.17 0.04 −1.15 2.83 P-ReA — 68.3— 1.68 — — ID = 3.88 mm 7.88 8.86 −2.30 0.34 −2.62 2.76 15.8 6.82 0.720.27 0.23 3.90 23.7 3.56 −2.99 0.178 −3.08 4.78 M-AsA — 97.5 — 0.21 — —ID = 1.20 mm 65.5 16.7 −2.93 0.17 1.83 2.54 131 6.94 −0.74 0.07 −2.803.59 196 1.36 0.91 0.04 −2.59 4.39

The magnitude and phase of each the fundamental and harmonic frequenciesof pressure and flow waveforms are quantified in Table 1 and are showngraphically in FIGS. 5a and 5b for a pig renal artery (P-ReA). Thevolumetric flow rates shown in Table 1 are calculated using the inverseWomersley method with measured flow velocities and the Fourier Analysis.Specifically, referring to FIGS. 5a and 5b , the composite hemodynamicpressure and flow waveforms from the pig renal artery are shown, where,in FIG. 5a , blood pressure is a measured quantity that is reconstructedusing a Fourier analysis, while in FIG. 5b , volumetric flow rate iseither provided by Doppler or calculated from the centerline velocity asdescribed. The composite waveforms (dotted lines) in FIG. 5a and FIG. 5bconsist of the mean value (n=0, solid line), fundamental frequencycomponent (n=1, open circle waveform), and the n=2 (open squarewaveform) and n=3 (open diamond waveform) harmonic frequencies shown inFIG. 5a for pressure and in FIG. 5b for volumetric flow. These compositewaveforms are similar to the superimposed waveforms shown in FIG. 1. TheReynolds number is a quantity that relates inertial to viscous effectsand is given by

$\begin{matrix}{\eta = \frac{2\; \overset{\_}{u}r_{i}\rho}{\mu}} & (15)\end{matrix}$

and all calculated Reynolds numbers yield laminar flow results evenduring peak flow confirming the laminar flow assumption.

Magnitude M_(n) and phase φ_(n) calculations of the mean frequency n=0,the fundamental frequency n=1, and the remaining harmonic frequenciesn=2 and n=3 demonstrate the dominance of the low and zero frequencyeffects on hemodynamics. Of note, a very good representation of bloodpressure and flow could be achieved with only the mean, fundamental, and1 or 2 of the harmonic frequencies. Low Womersley numbers were shown atlower frequencies in the smaller blood vessels that velocity profilestend towards Poiseuille-type parabolic flow.

The complete velocity profile across the radius of each blood vessel(a-c) can be seen in FIGS. 6a -6 c, where, specifically, FIGS. 6a-6cshow the velocity profiles (3D map) and measured centerline velocity(dotted line) for (a) Human Radial Artery, (b) Pig Renal Artery, (c) andMouse Aorta. For each sample, the centerline velocity is the onlymeasured velocity location based on the digitized literature andsuperimposed on the figure at r=0. The temporal and spatial differencesin velocity profile are apparent.

Turning now to FIGS. 7a -7 c, using equation (6) the mean (solid flatline) and pulsatile (solid parabolic/sinusoidal line) wall shearstresses (τ_(w)) are plotted for (a) Human Radial Artery, (b) Pig RenalArtery, (c) and Mouse Aorta for 2 seconds.

The best fit parameters of the 4-element Windkessel simulation, asconfigured in FIG. 4, are found in Table 2 and are set to minimize thedifference between the desired and actual pressure waveforms.

TABLE 2 Best fit parameters of the 4-element Windkessel model and thecumulative error for each of the mammalian blood vessels described inTable 1 $R_{1}\left( \frac{{mmHg} \cdot s}{ml} \right)$$R_{2}\left( \frac{{mmHg} \cdot s}{ml} \right)$$C\left( \frac{ml}{mmHg} \right)$$L\left( \frac{{mmHg} \cdot s^{2}}{ml} \right)$ e_(p) H-RaA 503 3920.0003 4.39 0.03 H-ReA 39.9 0.64 0.0010 10 0.10 M-AsA 485 0.56 0.00010.21 0.05

FIGS. 8a-8c demonstrate a plot of desired pressure, based on dataobtained from the literature, and the modeling results using the4-element Windkessel for each blood pressure. Specifically, the desiredpressure response is shown as a solid line and the simulated pressureresponse is shown as open circles pressure responses using the best fitparameters of the electro-hydraulic 4-element Windkessel model. Thedesired pressure is reported in the literature for (a) Human RadialArtery, (b) Pig Renal Artery, (c) and Mouse Aorta, which also shows azoomed-in section on one mouse cardiac cycle.

SUMMARY

Hemodynamic studies of the last 45 years have provided a wealth ofinformation that can be used as a baseline to design modern vascularculture systems. In this Example, contemporary blood flow studies weredigitized and analyzed using a Fourier transform approach to recreatecomplex waveforms as mathematical equations consisting of pulsatilepressure and centerline flow velocity. An inverse Womersley approach wasused to calculate the velocity profile, volumetric flow, and wall shearstress. A 4-element Windkessel model consisting of 2 resistors, acapacitor, and an inductor was used as a basis for hardware design usingthe electro-hydraulic analogy. The coefficients of the 4-ElementWindkessel were found by minimizing the error between the desired andsimulated blood pressure waveforms.

As a result of the data collected, the following statements provide aguideline for the construction of the pulsatile perfusion bioreactor ofthe present invention:

i. The volumetric flowrate q represents the output from a pump or pumps.The current configuration includes a single steady flow pump withconstant output equal to that of M^(q) ₀ and 3 peristaltic pumps thathave a sinusoidal output of magnitude M^(q) ₁₋₃ and phase φ^(q) ₁₋₃.Each of the peristaltic pumps would have rotational frequency of ω=n·ω.

ii. The value of resistors R₁ and R₂ represent the effect of adding arestriction to flow. For R₁, the most versatile and biocompatiblesolution would be to use an electronically controlled pinch valve. R₂,on the other hand, must also account for the resistance of the vesselitself so that the total value is R₂=(dp/dz)/q+R_(e) where R_(e)represents the extra resistance and may take a form similar to that ofR₁.

iii. The capacitance C is a combination of the compliance of the culturetubing and a compliance chamber. The tubing compliance is dependent uponlength, diameter, and material stiffness. The compliance chamber can beassembled with an adjustable volume V of compressible gas so thatC=dV/dp and the relationship between the volume and pressure for a gascan be found using the ideal gas law.

iv. The inductance L accounts for the inertia of the blood mass andtakes the form L=(ρ·Δl)/(πr_(i) ²). Since the radius of the vesselremains constant, a shorter or longer section of tissue may be used toadjust this parameter. Additionally, the length l of the vessel may alsobe limited experimentally so the density of the media can be alteredwith an additive to match the required value of inductance tocompensate.

v. A pressure transducer and flow velocity probe can be used to monitorhemodynamic waveforms. Simple versions, with limited risk forcontamination, include a manometer and a vascular Doppler probe.

EXAMPLE 2

Example 2 demonstrates the ability to culture an EBV in the pulsatileperfusion bioreactor of the present invention over a 10 day cultureperiod where the EBV was subjected to desired stress levels viaadjustment of the composite pressure and flow waveforms to which the EBVwas exposed, while also maintaining sterility and viability.

Pig tissue was obtained fresh from a local abattoir and dissections wereperformed immediately following tissue acquisition. Renal arteries werecarefully dissected from the surrounding tissue and mounted to ourperfusion device using 6.0 braided sutures. All processes occurred whilesubmerged in culture media. The supplemented growth media used in thedevice consists of Dulbecco's Modified Eagle's Media with 2% heatinactivated fetal bovine serum, 2% L-glutamine, 1000 units/L penicillin,and 1000 g/L streptomycin.

The capacitance was adjusted by changing the volume of air in thecompliance chamber. The magnitude and phase angle of each of thechannels of the pulsatile pump were adjusted according to Table 2 (ReA).Once the tissue was mounted in the culture chamber, it was placed insidethe incubator with the harsh environment camera and connected to thesupply and return tubing for both the lumen and reservoir. The LabViewprogram was initiated and the pump motor started under low-pressure,low-flow conditions to remove bubbles. Once bubbles were removed, thepressure control was initiated and culture commenced. A hypertensiveculture was also performed for 7 days (not shown) where the magnitude ofthe steady value of pressure control was 180 mmHg. Every two days for 10days the culture media was exchanged for fresh media.

At the end of the culture period, vessels were pressurized to 80 mmHgand viability was assessed using Phenylephrine (10⁻⁵ M) to elicit smoothmuscle contraction and carbamocholine chloride to induce nitric oxidedependent vasorelaxation (10⁻⁵ M). Contractility and dilation wereobserved for this example. Vessels were removed and prepared forpost-culture analysis.

These and other modifications and variations to the present inventionmay be practiced by those of ordinary skill in the art, withoutdeparting from the spirit and scope of the present invention, which ismore particularly set forth in the appended claims. In addition, itshould be understood the aspects of the various embodiments may beinterchanged both in whole or in part. Furthermore, those of ordinaryskill in the art will appreciate that the foregoing description is byway of example only, and is not intended to limit the invention sofurther described in the appended claims.

What is claimed:
 1. A pulsatile perfusion bioreactor for culturing oneor more engineered blood vessels having a lumen and a wall, thepulsatile perfusion bioreactor comprising: a chamber for holding theengineered blood vessel and cell culture media; a mechanical propertymonitoring system for measuring and controlling axial tensile stress,circumferential tensile stress, shear stress, or a combination thereofimparted on the wall of the engineered blood vessel and for measuringand controlling axial stretch, circumferential stretch, or a combinationthereof imparted on the wall of the engineered blood vessel; a pumpsystem for delivering cell culture media through the lumen of theengineered blood vessel, wherein the engineered blood vessel is exposedto a composite pressure waveform and a composite flow waveform as thecell culture media is delivered through the lumen, the pump systemcomprising a steady flow pump and a peristaltic pump, wherein thecomposite pressure waveform comprises a mean pressure component, a firstharmonic frequency pressure component, and a second harmonic frequencypressure component, and wherein the composite flow waveform componentcomprises a mean flow component, a first harmonic frequency flowcomponent, and a second harmonic frequency flow component; and acomputer interface for monitoring and adjusting the composite pressurewaveform, the composite flow waveform, or a combination thereof tomaintain a predetermined axial tensile stress level, a predeterminedcircumferential stress level, a predetermined shear stress level, or acombination thereof.
 2. The bioreactor as in claim 1, wherein thecomposite pressure waveform and the composite flow waveform are derivedfrom a pressure waveform and a flow waveform of a native blood vessel,wherein the engineered blood vessel is a replacement for the nativeblood vessel.
 3. The bioreactor as in claim 1, wherein the steady flowpump delivers the mean pressure component of the composite pressurewaveform and the mean flow component of the composite flow waveform. 4.The bioreactor as in claim 1, wherein the peristaltic pump delivers apulsatile flow of cell culture media through the lumen, wherein theperistaltic pump comprises a first pump head and a second pump head,wherein the first pump head provides the first harmonic frequencypressure component of the composite pressure waveform and the firstharmonic frequency flow component of the composite flow waveform, andwherein the second pump head provides the second harmonic frequencypressure component of the composite pressure waveform and the secondharmonic frequency flow component of the composite flow waveform.
 5. Thebioreactor as in claim 4, wherein the peristaltic pump further comprisesa third pump head, wherein the third pump head provides a third harmonicfrequency pressure component of the composite pressure waveform and athird harmonic frequency flow component of the composite flow waveform.6. The bioreactor as in claim 1, further comprising a compliancechamber.
 7. The bioreactor as in claim 1, further comprising a pressuretransducer.
 8. The bioreactor as in claim 1, further comprising astepper motor controlled pinch valve.
 9. The bioreactor as in claim 1,further comprising a camera for measuring the engineered blood vessellength, wall diameter, and wall thickness.
 10. The bioreactor as inclaim 1, wherein the chamber is located in an incubator.
 11. Thebioreactor as in claim 1, wherein the engineered blood vessel comprisesa natural material or a synthetic material.
 12. The bioreactor as inclaim 1, wherein the engineered blood vessel includes endothelial cells.13. The bioreactor as in claim 1, wherein the engineered blood vesselincludes smooth muscle cells.
 14. A method of culturing a one or moreengineered blood vessels having a lumen and a wall inside a pulsatileperfusion bioreactor, the method comprising: inserting the engineeredblood vessel to be cultured into a chamber; filling the chamber withcell culture media; delivering cell culture media through the lumen ofthe engineered blood vessel via a pump system, wherein the engineeredblood vessel is exposed to a composite pressure waveform and a compositeflow waveform as the cell culture media is delivered through the lumen,the pump system comprising a steady flow pump and a peristaltic pump,wherein the composite pressure waveform comprises a mean pressurecomponent, a first harmonic frequency pressure component, and a secondharmonic frequency pressure component, and wherein the composite flowwaveform component comprises a mean flow component, a first harmonicfrequency flow component, and a second harmonic frequency flowcomponent; measuring axial tensile stress, circumferential tensilestress, shear stress, axial stretch, circumferential stretch, or acombination thereof imparted on the wall of the engineered blood vesselvia a mechanical property monitoring system; monitoring and adjustingthe composite pressure waveform, the composite flow waveform, or acombination thereof to maintain a predetermined axial tensile stresslevel, a predetermined circumferential stress level, a predeterminedshear stress level, a predetermined axial stretch level, a predeterminedcircumferential stretch level, or a combination thereof via a computerinterface.
 15. The method as in claim 14, wherein the composite pressurewaveform and the composite flow waveform are derived from a pressurewaveform and a flow waveform of a native blood vessel, wherein theengineered blood vessel is a replacement for the native blood vessel.16. The method as in claim 14, wherein the steady flow pump delivers themean pressure component of the composite pressure waveform and the meanflow component of the composite flow waveform.
 17. The method as inclaim 14, wherein the peristaltic pump delivers a pulsatile flow of cellculture media through the lumen, wherein the peristaltic pump comprisesa first pump head and a second pump head, wherein the first pump headprovides the first harmonic frequency pressure component of thecomposite pressure waveform and the first harmonic frequency flowcomponent of the composite flow waveform, and wherein the second pumphead provides the second harmonic frequency pressure component of thecomposite pressure waveform and the second harmonic frequency flowcomponent of the composite flow waveform.
 18. The method as in any ofclaim 17, wherein the peristaltic pump further comprises a third pumphead, wherein the third pump head provides a third harmonic frequencypressure component of the composite pressure waveform and a thirdharmonic frequency flow component of the composite flow waveform. 19.The method as in claim 15, wherein the pulsatile perfusion bioreactorincludes a compliance chamber, wherein the compliance chamberfacilitates adjustment of the composite pressure waveform.
 20. Themethod as in claim 15, wherein pressure is measured via a pressuretransducer and a stepper motor controlled pinch valve is utilized toadjust resistance within the pulsatile perfusion bioreactor, whereinadjusting the resistance results in an adjustment to the pressure.